Introduction

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Liver damage involving centrilobular hepatocytes occurs as a result of exposure to 1,1-dichloroethylene (DCE), a chemical used extensively in the plastics manufacturing industry (Coleman et al., 1976; Forkert et al., 1986). It is also a degradation product of trichloroethylene and is found in conjunction with the latter as a water contaminant (Semprini, 1995). Metabolic studies have implicated covalent binding of DCE metabolites to cellular macromolecules as a mechanism responsible for the cytotoxic response. The extent of binding was dose-dependent and correlated with the severity of centrilobular necrosis (Forkert et al., 1986; Forkert and Moussa, 1991). Furthermore, immunohistochemical studies using an antibody that recognizes DCE protein adducts showed dose-dependent adduct formation that was preferentially localized in the centrilobular hepatocytes (Forkert, 1999). Subcellular fractions to which DCE was covalently bound were those from nuclei, cytosol, mitochondria, and microsomes (Okine et al., 1985; Okine and Gram, 1986). Other studies showed that DCE treatment produced morphological alterations primarily in sinusoidal and lateral plasma membranes, nuclei, and mitochondria (Reynolds et al., 1975). Early alterations involving mitochondria were manifested as swelling, disruption of cristae, and loss of mitochondrial matrix density (Reynolds et al., 1975; Reynolds and Moslen, 1977). Moreover, DCE treatment produced decreased histochemical staining of the inner mitochondrial membrane enzymes, succinate dehydrogenase and cytochrome oxidase. Staining for the outer mitochondrial membrane enzyme, monoamine oxidase, was also decreased (Chieco et al., 1982). These findings suggested that mitochondria are potential primary sites of damage in DCE-mediated hepatic injury.

The mechanism of DCE-induced hepatotoxicity involves cytochrome P450-dependent metabolism to reactive intermediates (Forkert et al., 1986, 1987). Subsequent studies in mice revealed that CYP2E1 is a major cytochrome P450 isoform catalyzing the oxidative metabolism of DCE (Kainz et al., 1993; Lee and Forkert, 1994). The primary metabolites formed from DCE oxidation in rat and murine liver microsomal incubations were the epoxide 2,2-dichloroacetaldehyde and 2-chloroacetyl chloride (Fig. 1) (Costa and Ivanetich, 1984; Liebler et al., 1985, 1988; Dowsley et al., 1995). The DCE epoxide was the major metabolite produced, whereas 2-chloroacetyl chloride was found at minimal levels (Dowsley et al., 1995). Acetal, the hydrate of 2,2-dichloroacetaldehyde, was also detected. The secondary metabolites generated included products of conjugation with glutathione (GSH) that were identified as 2-(S-glutathionyl)acetyl glutathione and 2-S-glutathionyl acetate (Fig. 1). These conjugates were major metabolites detected and were the products of conjugation of DCE epoxide with GSH. Because GSH depletion is associated with DCE-induced toxicity, it is plausible that the epoxide is responsible for mediating centrilobular necrosis by conjugating with GSH and depleting the GSH pool. This assumption is consistent with findings showing that rodents with reduced GSH levels due to fasting or pretreatment with diethylmaleate are more vulnerable to DCE-induced hepatotoxicity (Jaeger et al., 1974; Okine et al., 1985). Similarly, pretreatment with buthionine sulfoximine decreased GSH levels and exacerbated the toxicity of hepatocytes exposed to DCE in suspension (Kainz et al., 1993). These findings strongly supported GSH conjugation of DCE metabolites as a protective mechanism against DCE-induced hepatic injury.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Scheme of proposed pathway of DCE metabolism. The GSH conjugates are identified as follows: [A], S-(2,2-dichloro-1-hydroxy)ethyl glutathione; [B], 2-(S-glutathionyl)acetyl glutathione; [C], 2-S-glutathionyl acetate; and [D], S-(2-chloroacetyl)glutathione.

In the present study, our objective was to obtain evidence to demonstrate that mitochondria are an early primary target of DCE-induced hepatotoxicity. We have performed dose- and time-dependent studies in mice to determine the effects of DCE administration on mitochondrial respiratory status. Mitochondrial respiration was also investigated after pretreatment with N-acetyl-L-cysteine (NAC), an agent that increases the availability of intracellular GSH through provision of cysteine for GSH biosynthesis as well as functioning as an antioxidant (Corcoran et al., 1985; Cotgreave, 1997). Furthermore, all respiratory data were temporally correlated with hepatotoxicity.

	Materials and Methods

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Chemicals and Reagents. Chemicals and reagents were obtained from suppliers as follows: 1,1-dichloroethylene (>99% purity) (Aldrich Chemical Co., Montreal, QC, Canada); Bio-Rad protein assay dye reagent concentrate (Bio-Rad, Hercules, CA); and N-acetyl-L-cysteine (>99% purity), succinic acid (disodium salt), L-glutamic acid (monosodium salt), rotenone (95-98% pure), D-mannitol, HEPES, ADP (sodium salt), bovine serum albumin, -glutamyl glutamate, GSH, and t-butyl hydroperoxide (Sigma-Aldrich, St. Louis, MO). All other chemicals were of reagent grade and were obtained from standard commercial suppliers.

Animal Treatment. Female CD-1 mice (25-30 g) were obtained from Charles River Canada (St. Constant, QC, Canada). They were maintained on a 12-h light/dark cycle and given free access to food (Purina Rodent Chow; Ralston Purina International, Strathroy, ON, Canada) and drinking water. After acclimatization to laboratory conditions for at least 5 days, mice were randomly assigned to control or treatment groups. For dose-dependent assessment of respiratory status, mice were treated with DCE (50, 75, 100, 125, and 150 mg/kg i.p.) in corn oil and were sacrificed by cervical dislocation 2 h later. In time-course experiments, mice were treated with DCE (125 mg/kg i.p.) and sacrificed 10, 20, 30, 60, 90, and 120 min later. In studies that examined the effects of NAC (1200 mg/kg i.p. in distilled H₂O, pH 7.0) (Donnelly et al., 1993), NAC was administered 15 min before treatment with DCE (125 mg/kg), and mice were sacrificed 1 h later. For measurements of hepatic GSH content, mice were treated with DCE (125 mg/kg i.p.) and were sacrificed 10, 20, 30, 60, 120, 240, 360, and 480 min later. For measurements of serum alanine aminotransferase (ALT) activity, mice were treated with DCE (125 mg/kg) for 10, 20, 30 60, 90, 120, and 240 min. Mice were then anesthetized with sodium pentobarbital (120 mg/kg i.p.), and intracardiac blood was obtained for assessment of ALT activity. In all experiments, control mice were treated with equivalent volumes of the appropriate vehicle and were sacrificed at times corresponding to those in the experimental groups.

Isolation of Mitochondria. Mitochondria were isolated according to Johnson and Lardy (1967), with modifications. All procedures were carried out on ice or at 4°C. After cervical dislocation, livers were rapidly dissected into ice-cold isolation medium (0.25 M sucrose and 1 mM EDTA, pH 7.4). Gall bladders were removed and livers were blotted dry and weighed. Livers were then minced in fresh isolation medium and gently homogenized manually [10% (w/v) in isolation medium] using a Dounce homogenizer with a loose-fitting pestle. The resulting 10% homogenate was centrifuged at 500g for 10 min. The supernatant fraction was retained, whereas the pellet was washed with fresh isolation medium and recovered by centrifugation at 500g for 10 min. The supernatant fraction from this wash was combined with the initial supernatant fraction and again centrifuged at 500g for 10 min. The resulting supernatant fraction was centrifuged at 5000g for 15 min to obtain the mitochondrial pellet. This pellet was washed once with isolation medium and again with a respiration reaction buffer (70 mM sucrose, 220 mM mannitol, 2 mM HEPES, 0.5 mM EDTA, 2.5 mM MgCl₂, 0.5 mM KH₂PO₄, and 2 mM K₂HPO₄, pH 7.4), and the purified mitochondria were sedimented at 12,000g for 10 min. All washes were carried out in volumes amounting to 25% of the original homogenate with careful removal of lipid, which adhered to the walls of the centrifuge tubes. The final mitochondrial pellet was resuspended in respiration reaction buffer (1 ml/g tissue) to produce a suspension containing 25-40 mg of mitochondrial protein per milliliter. An aliquot was removed for determination of protein concentration by the Bradford (1976) method, using bovine serum albumin as the standard.

Polarographic Measurement of Oxygen Consumption. Oxygen consumption of isolated mitochondria was measured at 30°C using a model 5300 biological oxygen monitor and model 5331 Clarke-type polarographic oxygen probe (YSI, Inc., Yellow Springs, OH). Respiration buffer (3 ml), containing 10 mM glutamate, was added to a magnetically stirred sample chamber and allowed to equilibrate. Glutamate (complex I)-supported state 4 (resting) respiration was initiated by adding a 100-µl aliquot of the mitochondrial suspension (1.0-1.5 mg of protein) to the sample chamber. Subsequent addition of ADP (900 nmol in 50 µl of reaction medium) generated glutamate-supported state 3 (ADP-stimulated) respiration. After returning to state 4 respiration, glutamate-supported respiration was inhibited by adding rotenone (3 µM final concentration) to the sample chamber. Succinate (complex II)-supported state 3 and state 4 respiration was then assessed by consecutive addition of ADP (450 nmol in 25 µl of reaction medium) and succinate (10 µM final concentration). To assess the integrity of mitochondrial respiratory function, the respiratory control ratio (RCR, state 3/state 4) and ADP:O ratio were calculated (Estabrook, 1967).

Effects of in Vitro DCE Exposure on Mitochondrial Respiration. Isolated hepatic mitochondria were incubated with 1, 2, and 4 mM DCE at room temperature for 15, 30, and 60 min. Aliquots (100 µl) of the mitochondrial suspension were added to the sample chamber for analysis of rates of oxygen consumption. t-Butyl hydroperoxide (tBH; 1 mM), which has previously been shown to inhibit hepatic mitochondrial respiration (Lash et al., 1995), was used as a positive control in these mitochondrial toxicity assays.

Measurement of Hepatic GSH Content. Tissue GSH levels were measured by the method of Fariss and Reed (1987). Briefly, liver tissue samples (50-100 mg) were pulverized in liquid nitrogen and vortexed in 1 ml of 10% (v/v) perchloric acid containing 1 mM bathophenanthroline-disulfonic acid. Perchloric acid-insoluble material was removed by centrifugation (15,000g for 3 min), and 0.5 ml of the supernatant was added to a Wheaton vial containing 50 µl of 15 mM -glutamyl glutamate, which was used as the internal standard. After the addition of iodoacetic acid (50 µl, 100 mM in 0.2 mM m-cresol purple), the pH of the samples was adjusted to 8 to 9 with the addition of a potassium hydroxide/potassium bicarbonate solution (2 M KOH and 2.4 M KHCO₃). The samples were then mixed and incubated for 10 min in the dark. Subsequently, 1 ml of 1% 1-fluoro-2,4-dinitrobenzene was added to the samples that were then capped, vortexed, and stored overnight at 4°C. High-performance liquid chromatography analysis was conducted using an aminopropyl silica ion-exchange column (5 µm, 4 × 250 mm; SGE International, PTY Ltd., Ringwood, Australia). Initially, 4 parts of mobile phase A (80% methanol), and 1 part of mobile phase B (0.5 M ammonium acetate in 64% methanol) was allowed to equilibrate at a flow rate of 0.75 ml/min. After a 100-µl injection of the derivatized sample, increasing concentrations of mobile phase B (20-99%) were introduced to the column over a period of 10 min to elute the chromophore derivative. The derivatized concentrations of -glutamyl glutamate and GSH were determined by detection at 360 nm.

Measurement of ALT Activity. Serum ALT activity was measured by the method of Bergmeyer et al. (1978) using an ALT 20 kit (Sigma Diagnostics, St. Louis, MO). ALT activity was determined at 10, 20, 30, 60, 90, 120, and 240 min after DCE (125 mg/kg) treatment.

Statistical Analysis. Data are expressed as mean ± S.D. Data were analyzed by one-way analysis of variance followed by Tukey's test to determine significant differences between experimental groups (p < 0.05).

	Results

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Effects of in Vivo DCE Treatment on Mitochondrial Respiration. The effects of DCE on mitochondrial respiration were examined in dose- and time-dependent studies to identify early alterations in mitochondrial function in the toxic response. In dose-dependent studies, a decreasing trend was observed in state 3 respiration rates for glutamate (complex I)- and succinate (complex II)-supported respiration over the range of DCE doses administered. A DCE dose of 125 mg/kg was found to significantly inhibit glutamate- and succinate-supported state 3 respiration rates by 42 and 40%, respectively (Figs. 2A and 3A). In contrast, no significant changes in state 4 respiration rates were observed at any dose for either substrate (Figs. 2B and 3B). Glutamate- and succinate-supported respiration was found to be completely uncoupled with DCE doses of 150 mg/kg or higher (data not shown). In time-dependent studies, a marked decline in glutamate- and succinate-supported state 3 respiration rates was followed by a more level phase. State 3 respiration rates for glutamate-supported respiration were significantly decreased from 20 to 120 min after DCE (125 mg/kg) treatment, whereas those for succinate-supported respiration were significantly decreased from 90 to 120 min (Figs. 4A and 5A). No significant changes in state 4 respiration rates were observed at any time for either substrate (Figs. 4B and 5B).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Dose-dependent effects of DCE treatment (2 h) on glutamate-supported (complex I) mitochondrial respiration. Respiration was measured as state 3 (A) and state 4 (B) rates of oxygen consumption. RCR (C; state 3/state 4) and ADP:O (D) values were then calculated. Data for the control are depicted at time 0. Data are expressed as mean ± S.D. of measurements from three separate experiments. , significantly different from the control (p < 0.001).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Dose-dependent effects of DCE treatment (2 h) on succinate-supported (complex II) mitochondrial respiration. Respiration was measured as state 3 (A) and state 4 (B) rates of oxygen consumption. RCR (C; state 3/state 4) and ADP:O (D) values were then calculated. Data for the control are depicted at time 0. Data are expressed as mean ± S.D. of measurements from three separate experiments. , significantly different from the control (p < 0.001).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Time-dependent effects of 125 mg/kg DCE treatment on glutamate-supported (complex I) mitochondrial respiration. Respiration was measured as state 3 (A) and state 4 (B) rates of oxygen consumption. RCR (C; state 3/state 4) and ADP:O (D) values were then calculated. Control mice were treated with corn oil. Data are expressed as mean ± S.D. of measurements from three separate experiments. , significantly different from the controls (p < 0.001).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Time-dependent effects of 125 mg/kg DCE treatment on succinate-supported (complex II) mitochondrial respiration. Respiration was measured as state 3 (A) and state 4 (B) rates of oxygen consumption. RCR (C; state 3/state 4) and ADP:O (D) values were then calculated. Control mice were treated with corn oil. Data are expressed as mean ± S.D. of measurements from three separate experiments. , significantly different from the controls (p < 0.001).

Effects of in Vitro DCE Exposure on Hepatic Mitochondria. Isolated hepatic mitochondria were incubated with 1, 2, and 4 mM DCE for 15, 30, and 60 min to determine whether the parent compound, exclusive of its P450-derived metabolites, produced mitochondrial perturbations. No effects on glutamate- or succinate-supported state 3 and state 4 respiration were observed, regardless of DCE concentration or incubation time; as a result RCRs and ADP:O ratios remained unaffected (data not shown). In contrast, 1 mM tBH inhibited glutamate-supported state 3 respiration rates by 32, 45, and 65% at 15, 30, and 60 min, respectively. Succinate-supported state 3 respiration rates were inhibited by 27, 48, and 64% at 15, 30, and 60 min, respectively. Also, at these time-points, glutamate-supported state 4 respiration rates were increased by 14, 20, and 22%, and succinate-supported state 4 respiration rates were increased by 9, 15, and 24%, respectively. As a consequence of tBH-induced alterations in state 3 and state 4 respiration rates, the control RCR (3.7 ± 0.4) for glutamate oxidation was decreased to 2.2 ± 0.3, 1.7 ± 0.4, and 1.2 ± 0.2 at 15, 30, and 60 min, respectively. The control RCR (3.3 ± 0.2) for succinate oxidation was decreased to 2.5 ± 0.4, 1.7 ± 0.3, and 1.2 ± 0.3 at 15, 30, and 60 min, respectively.

Effects of DCE and/or NAC on GSH Content. To identify a potential relationship between DCE-induced mitochondrial disturbances and hepatic GSH levels, GSH levels were measured at 10 to 480 min after treatment with 125 mg/kg DCE or the vehicle (Fig. 6). GSH levels were significantly reduced at 30 and 60 min by 28 and 37%, respectively. Thereafter, GSH levels gradually increased and at 4 h returned to control levels. Additionally, GSH levels were determined at 1 h in mice treated with 1200 mg/kg NAC or 125 mg/kg DCE and NAC. GSH levels were similar in control mice and mice treated with NAC or DCE and NAC (Fig. 7).

View larger version (42K): [in this window] [in a new window]   Fig. 6.   Time course of alterations in tissue GSH in livers of control and 125 mg/kg DCE-treated mice. Control mice are treated with corn oil. Data are expressed as mean ± S.D. of measurements from three separate experiments. , significantly different from the controls (p < 0.001).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Liver glutathione levels in control, NAC-, DCE-, and NAC + DCE-treated mice. Mice were treated with 1200 mg/kg i.p. NAC or the vehicle at 15 min before treatment with 125 mg/kg i.p. DCE or corn oil, and sacrificed 1 h later. Data are expressed as mean ± S.D. of measurements from three separate experiments. , significantly different from the control (p < 0.001).

Effect of NAC on Mitochondrial Dysfunction. To further investigate the early effects of DCE treatment on hepatic mitochondrial respiration, 1200 mg/kg NAC was administered 15 min before 125 mg/kg DCE and the respiratory status was evaluated. In preliminary experiments, it was determined that NAC alone had no effect on any of the respiratory parameters assessed. DCE-induced inhibition of glutamate- and succinate-supported state 3 respiration rates was diminished by pretreatment with NAC (Fig. 8A). State 3 respiration rates for mice treated with DCE and NAC were less than the controls and were significantly higher than in mice treated with DCE alone. Glutamate- and succinate-supported state 4 respiration rates were similar for all treatment groups (Fig. 8B). Additionally, NAC treatment also decreased inhibition of RCRs and ADP:O ratios for glutamate and succinate oxidation (Fig. 8, C and D).

View larger version (37K): [in this window] [in a new window]   Fig. 8.   Effect of 125 mg/kg DCE treatment on glutamate (complex I)- and succinate (complex II)-supported respiration after pretreatment with 1200 mg/kg NAC. Mice were treated with NAC or the vehicle 15 min before DCE treatment and sacrificed 1 h later. The following respiratory parameters were then measured: state 3 respiration (A), state 4 respiration (B), respiratory control ratio (C), and ADP:O ratio (D). Data are expressed as mean ± S.D. of measurements from three separate experiments. a and c, significantly different from the corresponding controls (p < 0.001). b and d, significantly different from DCE alone (p < 0.001).

Measurement of ALT Activity. The hepatotoxic effect of DCE was observed over time by measurement of serum ALT activity. ALT activity was found to be significantly elevated at 2 h after DCE administration and was further increased after 4 h (Fig. 9). The effectiveness of NAC in protecting against DCE-induced hepatotoxicity was also investigated by measuring serum ALT activity. Pretreatment with NAC did not produce an increase in serum ALT activity (8.7 ± 2.7 U/l), compared with the control (4.2 ± 0.8 U/l).

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Time course of serum ALT activity after 125 mg/kg DCE treatment. Control mice were treated with corn oil. Data are expressed as mean ± S.D. of measurements from three separate experiments. , significantly different from the control (p < 0.001).

	Discussion

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The liver has been identified as a major target of DCE-induced cytotoxicity. Yet, despite a well characterized hepatic lesion, the early events critical to the onset of toxicity remained unclear. Morphological, histochemical, and covalent binding studies have implicated mitochondria in the early pathogenesis of DCE-mediated hepatotoxicity (Reynolds et al., 1975; Reynolds and Moslen, 1977; Chieco et al., 1982; Okine et al., 1985; Okine and Gram, 1986); however, biochemical data are lacking in this respect. In this study, we examined the early effects of DCE on respiratory parameters indicative of mitochondrial integrity and function. Isolated mouse liver mitochondria were used to examine the effects of DCE treatment on mitochondrial respiration. Dose-response experiments were initially performed to establish a dose that produced early, moderate inhibition of respiratory function at 2 h, a time point at which alterations in mitochondrial morphology were manifested (Reynolds et al., 1975; Kanz and Reynolds, 1986). A DCE dose of 125 mg/kg significantly reduced glutamate- and succinate-supported state 3 respiration rates as well as RCRs, and ADP:O ratios (Figs. 2 and 3). As a result, this DCE dose was used in subsequent studies. To evaluate the early effects of DCE on mitochondrial function, respiratory parameters were assessed in time-course experiments. State 3 respiration rates and RCRs for glutamate-supported respiration were significantly decreased as early as 20 min after DCE treatment, whereas those for succinate-supported respiration were significantly decreased at 90 min (Figs. 4 and 5). These findings are consistent with results of previous studies (Jaeger, 1977) showing a significant reduction in pyruvate- and malate-supported state 3 respiration in murine liver 1 h after inhalation exposure of DCE. To further assess the effects of DCE on mitochondrial function, the ADP:O ratio was determined and used as an index of oxidative phosphorylation. The ADP:O ratio reflected how efficiently mitochondria used the molecular substrate O₂ for flavoprotein-mediated ATP synthesis (Toyomizu et al., 1992). As early as 60 min after DCE treatment, the ADP:O ratio for glutamate oxidation was significantly decreased (Fig. 4). Succinate oxidation was not affected as rapidly as was glutamate oxidation, and the ADP:O ratio was significantly reduced by 90 min (Fig. 5). Hence, significant decreases in oxidative phosphorylation, as assessed by ADP:O ratios, were noted at 60 to 90 min after DCE treatment. The functional alterations in the mitochondria seemed to precede hepatic necrosis, as assessed by measurement of serum ALT activity. Serum ALT activity was significantly elevated at 2 h, whereas mitochondrial perturbations were evident as early as 20 min after DCE exposure (Figs. 4 and 9). Additionally, centrilobular necrosis was observed 24 h after treatment of mice with 125 mg/kg DCE (Forkert et al., 1986). Moreover, it has been reported that decreases in histochemical staining of mitochondrial membrane enzymes preceded increases in serum transaminase activities, a biomarker of hepatic injury (Chieco et al., 1982). Thus, these data suggested that mitochondrial alterations are associated with the initiation and/or early development of DCE-induced hepatotoxicity.

The time-dependent alterations of mitochondrial function produced by DCE have provided some insight into the sequence of events leading to the toxic outcome. Initial decreases of state 3 respiration rates without concomitant decreases of state 4 respiration rates suggested specific inhibition of enzymes in the respiratory chain proper and/or inhibition of utilization of nucleotides (i.e., the adenine nucleotide translocase). Further investigation, however, is required to discern the precise site(s) of inhibition. At times subsequent to 120 min after DCE treatment, oxidation in the respiratory chain was found to be uncoupled from ADP phosphorylation. As a result, respiration became uncontrolled and the concentration of ADP or P_i was no longer rate limiting (data not shown). Thus, complete uncoupling of oxidative phosphorylation occurred subsequent to inhibition of specific components of the respiratory chain.

Because DCE is readily metabolized in target tissues, it was of interest to determine whether the parent compound or its metabolites were associated with the observed mitochondrial dysfunction. To this end, isolated liver mitochondria were incubated with 1, 2, and 4 mM DCE for 15, 30, and 60 min, and mitochondrial respiration was assessed. Inhibition of respiratory parameters was not identified under these conditions. To validate the assay for mitochondrial toxicity in vitro, isolated mitochondria were incubated with tBH, a known mitochondrial toxicant, and this system was used as a positive control (Lash et al., 1995; Cawthon et al., 1999). tBH significantly inhibited state 3 respiration rates and increased state 4 respiration rates, resulting in significantly reduced RCRs. Hence, the in vivo metabolism of DCE seemed to be a necessary event in the development of mitochondrial toxicity.

In the present study, DCE-induced functional alterations in hepatic mitochondria coincided with changes in cellular GSH levels. Both the impairment of mitochondrial respiration and decreased GSH levels occurred rapidly and were maximal at approximately 1 h after DCE exposure (Fig. 6). However, whereas GSH levels were fully restored by 4 h, mitochondrial uncoupling was still evident at this time (data not shown). Thus, a relationship between mitochondrial dysfunction and hepatic GSH levels seemed to exist in the early time period after DCE treatment. To investigate the relationship between mitochondrial alterations, GSH and hepatotoxicity, respiration, GSH levels, and ALT activity were assessed after NAC pretreatment. Our results revealed that GSH levels were similar in control mice and mice treated with DCE and NAC (Fig. 7). Thus, NAC in providing cysteine for GSH synthesis, increased GSH levels and abrogated GSH diminution by DCE. Pretreatment with NAC inhibited both the impairment of mitochondrial respiration observed at 1 h after DCE treatment (Fig. 8) and the increase in serum ALT activity seen at 2 h. This association between early mitochondrial alterations and decreased GSH levels suggested that DCE-mediated GSH depletion may play a role in the onset of mitochondrial dysfunction and subsequent hepatotoxicity.

Although our data indicated that mitochondrial dysfunction is an early event in the development of DCE-induced hepatotoxicity, the precise underlying mechanism remains unclear. It is conceivable that DCE-derived metabolites arylate and/or oxidize essential protein sulfhydryls in the mitochondrial respiratory chain, thereby disrupting cellular energy homeostasis. Indeed, it has been shown previously that DCE metabolites, including the epoxide, conjugated readily in vivo and in vitro with GSH, the most abundant intracellular nonprotein sulfhydryl (Dowsley et al., 1995; Forkert, 1999). Alternately, oxidation of protein sulfhydryls may occur but seems a less likely occurrence because alterations to mitochondrial function outlast GSH recovery. Because GSH is the most abundant intracellular antioxidant, after its recovery, mitochondrial perturbations resulting from oxidation might be expected to recuperate. However, this response may not occur if the damage to mitochondria is sufficiently severe so that recovery is obviated. Both NADH dehydrogenase and succinate dehydrogenase contain several cysteine-rich iron-sulfur clusters that function in electron transport (Hatefi, 1985; Murray et al., 2000), and the activity of both enzyme complexes in this study has been shown to be inhibited. Decreased ATP production, subsequent to inhibition of these enzymes, is likely to have deleterious effects on numerous cellular functions, including Ca²⁺ homeostasis and maintenance of plasma membrane integrity (Dills and Klaassen, 1986). Such alterations may lead to cellular necrosis. However, there is currently no evidence to support a DCE-mediated mechanism of ATP depletion, and further investigation is warranted.

In conclusion, our results demonstrated that DCE-induced mitochondrial dysfunction occurs early in the toxic response and is inhibited under experimental conditions that prevent depletion of intracellular GSH. Thus, these data suggested that GSH plays an important role in conjugation and detoxification of DCE metabolites that might otherwise elicit mitochondrial toxicity.